The Study On Identification And Biological Characteristics Of Cancer Stem Cells Of Laryngeal Carcinoma

Laryngeal carcinoma(LC) is a common malignant disease in northeast area of China. Epidemiologic studies show that the number of patients is slightly increasing. Although improvements in the treatment of early and advanced carcinomas of larynx have been made in the past decades, the typical 5- year survival rates for all stages of disease are still in range of 62% to 67%. Mortality from this disease remains high because of the development of local and regional recurrences and metastasis. Therefore, it is essential for us to develop a deeper understanding of the biology of this disease to develop more effective therapies. In recent years, there has been overwhelming evidence in some malignancies supporting the notion that tumors contain cellular heterogeneity. The capability to sustain tumor formation and growth resides in a small proportion of tumor cells termed cancer stem cells or tumor-initiating cell. Such cells possess extensive proliferative and self-renewal potential and are responsible for maintaining the tumor clone. The discovery of cancer stem cells (CSCs) has played a pivotal role in changing our view of carcinogenesis .Tumor can be considered stem disease and cancer stem cells are thought to be responsible for tumor initiation , maintenance and growth. Growing evidence suggests cancer stem cells uniquely propagate many malignancies and are resistant to conventional therapies . Targeting these cells is essential for chronic and curative therapy .Recent studies have identified the existence of highly tumorigenic, self-replicating cancer stem cells in blood and solid tumors, including breast, brain, lung, prostate and pancreatic cancer through an experimental strategy that combines the sorting of tumor cell subpopulations with functional transplantation into appropriate animal models. The different cell surface phenotypes prospectively identify tumorinitiating subpopulations in solid tumors and even cell lines derived from tumors. It has not been reported about isolation and identification of cancer stem cells in laryngeal carcinoma until now. The purpose of this study was to identification and investigate the biological characteristics of cancer stem cells in laryngeal carcinoma with both in vitro and in vivo analyses.Part I : Preliminary study on subpopulation cell isolation and heterogeneity characterization of human laryngeal carcinomaObjective To isolate and characterize the heterogeneous subpopulation cells from human laryngeal carcinoma,and to explore the mechanism of heterogeneity in laryngeal carcinoma preliminarily.Methods Laryngeal carcinoma cells were cultured in vitro by primary tissue culture technique.Heterogeneous subpopulations were isolated by limiting dilution and were compared respectively on their functional status,proliferation capability ,cell surface antigen expression and growth condition in serum-free RPMI1640 medium.Each subpopulation was implanted in nude mice subcutaneously to measure the tumor forming ability.Results Two different subpopulations of typeⅠand typeⅡlaryngeal carcinoma cells were derived from primary tumor tissues.TypeⅠcells are multangular in shape with protuberances and could produce new tumor.However,typeⅡcells show a spindle feature and could not form new tumor when implanted in nude mice.Compared with typeⅡcells,typeⅠcells contained much less RNA and more G0/G1 cells,demonstrating increased proliferation capacity ,expressing CD44 and CD133 cell surface antigen, growing in serum-free RPMI1640 medium like tumor spheres.Furthermore,typeⅠcells can create heterogeneous subclone offspring,but typeⅡcells can not generate offspring cells . Conclusions Laryngeal carcinoma cells are heterogeneous with respect to characteristics on morphology,proliferation,functional and growth status.TypeⅠcells possess some characteristics of tumor stem cells.PartⅡ: The Study On Identification and Biological Characteristics of Cancer stem Cells in Laryngeal CarcinomaObjective To identify and study biological characteristics of cancer stem cells in laryngeal carcinoma . Methods Taking CD44 molecule as a marker to isolate CD44+ and CD44- subpopulation cells from laryngeal carcinoma cells. CD44+ and CD44- cells were compared on their degree of differentiation,proliferation capability and rate of forming clone . CD44+, CD133+, CD44+CD133+ and CD44+CD133- subset cells were isolated from laryngeal carcinoma cells by using flow cytometry for further study. To study the tumorigenic effects of different populations ,we developed a nude mouse tumor xenograft model. Mice were transplanted into the left flank at the injection dose 1×106,1×105,1×104,1×103 respectively. The mice were monitored for palpable tumor formation and were sacrificed after 4 weeks to assess tumor formation rate, volume and weight in vivo. Cell proliferation was analyzed by reading ultraviolet absorption spectrums at a wavelength of 490 nm on a Versamax microplate reader and soft agar clone forming rate was performed to detect tumorigenic ability in vitro. Boyden chamber migration assay was used to study the migration ability and immunochemistries for stem cell antigen Sca-1 andβ1-integrin were performed in highly tumorigenic and control cells . Isolated highly tumorigenic cells were seeded into serum-free media and the growth state of the cells were observed every 2 days .To observe differentiation ability , the spherical clone cells were cultured in serum media and detected about the offspring cells immigration .Semi-quantities RT-PCR and Western blot analyses were used to detect the expression levels of Bmi-1 in the different subpopulation cells .Results CD44+ tumor cells had stronger clone forming ability and expressed more CK14 protein and less Involucrin protein than CD44-cells. The percentages of CD44+ cells were about49.8%～53.5% and the median was 51.3%. After culturing CD44+ cells isolated from laryngeal carcinoma could proliferate and the percentage of CD44+ remained the same. Examination of subcutaneous tumors in nude mice showed that CD44+CD133+ cells generated a tumor with 1×103 cells ,and at the same injection dose (1×106 cells),both the average weights and volumes of the tumors were higher than those of other groups. Boyden chamber migration assay indicated that the cells number with invasion ability in CD44+CD133+ cells were significantly higher than other subset cells. The results of immunochemical analyses showed that stem cell antigen Sca-1 andβ1-integrin expression in CD44+CD133+ cells is abundant. CD44+CD133+ cells grown in serum-free conditions became clusters.As days passed, the size of cell clusters formed the spherical clone cells as some malignant brain tumors cells or pancreatic cancer cells did. Compared with other subset cells , CD44+CD133+ cells were demonstrated highly proliferation capacity and tumorigenic ability in vitro. Semi-quantitative RT-PCR and Western blot analyses provided strong evidence that Bmi-1 expression in CD44+CD133+ and CD133+ cells different from CD44+, CD44+CD133- and control cells remarkably.Conclusions Taken together, it was confirmed that CD44+CD133+ subset cells in laryngeal carcinoma posses some biological characteristics of tumor stem cells : CD44+CD133+ cells presented clone generative, self renewal, extensive differentiation ability and higher proliferative potential, which may be considered as cancer stem cells in laryngeal carcinoma.PartⅢCorrelation study of Bmi-1 expression in laryngeal carcinoma and construction of double-promoter pFIV–H1/U6 Bmi-1 siRNA recombinant vectorObjective To study Bmi-1 expression and clinicopathologic characteristics in laryngeal carcinoma and to construct double-promoter pFIV–H1/U6 recombinant plasmid expressing siRNA-Bmi-1.Methods The expression of Bmi-1 mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) in cancer samples and adjacent non-cancerous samples from 20 laryngeal carcinoma patients. The expression of Bmi-1 protein was examined by immunohistochemistry in archival paraffin-embedded tissue speciments. Statistical analysis was performed to investigate the association between the clinicopathologic features and Bmi-1 expression. Using human Bmi-1 gene sequences from Genbank, we designed and synthesized four pairs oligonucleotides as DNA template encoding siRNA-Bmi-1, cloning siRNA template into double-promoter pFIV-H1/U6 siRNA vector. And then we identified clones with the target siRNA template by using BamHI enzyme to digest PCR product. Results RT-PCR demonstrated that the levels of Bmi-1 mRNA in laryngeal carcinoma samples were significantly higher than those in adjacent non-cancerous tissues. Immunohistochemical staining showed that Bmi-1 was moderately or highly expressed in 67.39% of laryngeal carcinoma. Expression of Bmi-1 was highly correlated with clinical classification, lymph node metastasis and tumor differentiation(p<0.05).On the other hand, it was not correlated with gender, age or clinical type(p>0.05).PCR product from clones without an insert was be digested ,resulting in two bands at 81 and 91bp. Product frome positive clones was not be digested and the resulting band was 163 bp for siRNA templates.Conclusions Bmi-1 was overexpressed in laryngeal carcinoma. It was the foundation that construction of double-promoter H1/U6 siRNA-Bmi-1 vector for the following transfection and analysis of silenceing efficiency.